The present invention relates to proteins and peptides derived from a protein called Endothelial Cell Specific Molecule -1 (ESM-1).
It relates moreover to the use of such proteins and peptides in the treatment and diagnosis of diseases linked to leukocyte migration, and in particular to inflammatory diseases.
Positioned at the interface between circulating cells and tissues, the endothelial cells play a critical role in the homing and the local accumulation of leukocytes. Initial tethering and rolling, subsequent arrest and adhesion, and transendothelial migration constitute the current view of leukocyte migration. Leukocyte migration involves signal molecules, including selecting, chemoattractants, and integrins, which are present on endothelial cells. Their display of signals is carefully under the control of cytokines; E-selection, vascular cell adhesion molecule-1, IL-8; and RANTES (Regulated on Activation Normal T Cell Expressed) are expressed on endothelial cells only activated by cytokines, whereas ICAM-1, ICAM-2, AND IL-6, which are expressed constitutively in a low rate, are highly induced on endothelial cells in the presence of cytokines.
Vascular endothelium shows diversity among tissues, and there are fewer known mechanisms that regulate leukocyte migration and localization within specific tissues. E-selectin, GlyCAM-1, CD34, and MadCAM-1 contribute to the tissue-specific homing of circulating T-lymphocytes in skin, lymph nodes, and Peyer patches, respectively. GlyCAM-1, CD34, and MadCAM-1 are mucin-like carriers of selectin ligands. CD34 and MadCAM-1 are type 1 membrane glycoproteins, but GlyCAM-1 is secreted by the high endothelial veinules. In the other tissues, very little is known about the presence of such homing molecules on the endothelial cells. Therefore, identification of tissue- and endothelical cell-restricted molecules may contribute to a better understanding of these tissue specific leukocyte-endothelial cell interactions and to find some new method of therapy of diseases related with the leukocyte migration, in particular inflammatory diseases.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al., (1989); Glover, (1985); Gait, (1984); Hames and Higgins, (1985); Freshney, (1986); Perbal, (1984); and F. Ausubel et al., (1989).
Therefore, if appearing herein, the following terms shall have the definitions set out below.
The term xe2x80x9cisolatedxe2x80x9d for the purposes of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally present).
For example, a polynucleotide present in the natural state in a plant or an animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted in the genome of the plant or animal is considered as being xe2x80x9cisolatedxe2x80x9d.
Such a polynucleotide may be included in a vector and/or such a polynucleotide may be included in a composition and remains nevertheless in the isolated state because of the fact that the vector or the composition does not constitute its natural environment.
The term xe2x80x9cpurifiedxe2x80x9d does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.
A polynucleotide is in the xe2x80x9cpurifiedxe2x80x9d state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.
For the purposes of the present description, the expression xe2x80x9cnucleotide sequencexe2x80x9d may be used to designate either a polynucleotide or a nucleic acid. The expression xe2x80x9cnucleotide sequencexe2x80x9d covers the genetic material itself and is therefore not restricted to the information relating to its sequence.
The terms xe2x80x9cnucleic acidxe2x80x9d, xe2x80x9cpolynucleotidexe2x80x9d, xe2x80x9coligonucleotidexe2x80x9d or xe2x80x9cnucleotide sequencexe2x80x9d cover RNA, DNA, gDNA or cDNA sequences or alternatively RNA/DNA hybrid sequences of more than one nucleotide, either in the single-stranded form or in the duplex, double-stranded form.
A xe2x80x9cnucleic acidxe2x80x9d is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence or coding sequence.
The term xe2x80x9cnucleotidexe2x80x9d designates both the natural nucleotides (A, T, G, C) as well as the modified nucleotides that comprise at least one modification such as (1) an analog of a purine, (2) an analog of a pyrimidine, or (3) an analogous sugar, examples of such modified nucleotides being described, for example, in the PCT application No. WO 95/04 064.
xe2x80x9cIsolated polypeptidexe2x80x9d or xe2x80x9cisolated proteinxe2x80x9d is a polypeptide or protein which is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). xe2x80x9cIsolatedxe2x80x9d is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
Polypeptides of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a polypeptide of SEQ ID NO2 or SEQ ID NO3 according to the invention including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations are not expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
Lys for Arg and vice versa such that a positive charge may be maintained;
Glu for Asp and vice versa such that a negative charge may be maintained;
Ser for Thr such that a free xe2x80x94OH can be maintained; and
Gln for Asn such that a free CONH2 can be maintained.
A xe2x80x9cvectorxe2x80x9d is a replicon, such as plasmid, virus, phage or cosmid, to which another DNA segment-may be attached so as to bring about the replication of the attached segment. It is a circular or a linear DNA or RNA molecule which is either double-stranded or single-stranded. A xe2x80x9crepliconxe2x80x9d is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
Identity refers to sequence identity between two peptides or between two nucleic acid molecules. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical aminoacids at positions shared by these sequences. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a xe2x80x9ccomparison windowxe2x80x9d to identify and compare local regions of sequence similarity. A xe2x80x9ccomparison windowxe2x80x9d, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1972), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Solftware Package Release 7.0, Genetics. Computer Group, 575, Science Dr. Madison, Wis.), or by inspection. The best alignment (i.e., resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected. The term xe2x80x9csequence identityxe2x80x9d means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term xe2x80x9cpercentage of sequence identityxe2x80x9d is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, U or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
Summary of the Invention
According to the invention, a polypeptide or a protein having at least 85% amino acid identity as regards a reference polypeptide or protein possesses preferably at least 90%, more preferably at least 95%, 96%, 97%, 98%, 99%, 99.5% and most preferably at least 99.7% amino acid identity as regards the reference polypeptide or protein.
In a most preferred embodiment, a polypeptide having at least 85% amino acid identity with an ESM-1 protein of SEQ ID no2 or SEQ ID NO3 possesses the same biological properties or activities as regards the parent protein.
Most preferably, a protein having at least 85% amino acid identity with the secreted ESM-1 protein of SEQ ID NO3 possesses an ability to bind to the LFA-1 molecule of the same order of magnitude as the secreted ESM-1 protein itself. For example, a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% aminoacid identity with the secreted ESM-1 protein of SEQ ID NO3 is encompassed by the present invention if it binds to the LFA-1 molecule in a BIAcore system used in the experimental conditions disclosed in Example 10 with a dissociation constant comprised between 1 nM and 100 nM, advantageously between 1 nM and 50 nM, more preferably between 5 nM and 30 nM and most preferably between 8 nM and 25 nM.
It is therefore an object of the present invention to provide proteins and peptides which can be used in a method of therapy of diseases related with leukocyte migration.
It is another object of the present invention to provide S a method for determining the ESM-1 protein in biological samples.
In accomplishing these and other objects, there have been provided, in accordance with one aspect of the present invention, a protein the sequence of which shows an identity of at least 40, preferentially 60 or 70, and more preferentially 85% with a part of at least 25, 50, preferentially 70 aminoacids of the following sequence SEQ ID O 2.
Met Lys Ser Val Leu Leu Leu Thr Thr Leu Leu Val Pro Ala His Leu Val Ala Ala Trp Ser Asn Asn Tyr Ala Val Asp Cys Pro Gln His Cys Asp Ser Ser Glu Cys Lys Ser Ser Pro Arg Cys Lys Arg Thr Val Leu Asp Asp Cys Gly Cys Cys Arg Val Cys Ala Ala Gly Arg Gly Glu Thr Cys Tyr Arg Thr Val Ser Gly Met Asp Gly Met Lys Cys Gly Pro Gly Leu Arg Cys Gln Pro Ser Asn Gly Glu Asp Pro Phe Gly Glu Glu Phe Gly Ile Cys Lys Asp Cys Pro Tyr Gly Thr Phe Gly Met Asp Cys Arg Glu Thr Cys Asn Cys Gln Ser Gly Ile Cys Asp Arg Gly Thr Gly Lys Cys Leu Lys Phe Pro Phe Phe Gln Tyr Ser Val Thr Lys Ser Ser Asn Arg Phe Val Ser Leu Thr Glu His Asp Met Ala Ser Gly Asp Gly Asn Ile Val Arg Glu Glu Val Val Lys Glu Asn Ala Ala Gly Ser Pro Val Met Arg Lys Trp Leu Asn Pro Arg
Such a protein comprises preferentially between almost 40 and 200, more preferentially between almost 80 and 150 aminoacids.
Preferentially, at least a part of the sequence of this protein shows a similarity of at least 60, 75 and more preferentially 90% with a part of at least 50, preferentially 70 aminoacids of the sequence SEQ ID NO2.
Such a protein can show the following sequence SEQ ID NO 3.
Trp Ser Asn Asn Tyr Ala Val Asp Cys Pro Gln His Cys Asp Ser Ser Glu Cys Lys Ser Ser Pro Arg Cys Lys Arg Thr Val Leu Asp Asp Cys Gly Cys Cys Arg Val Cys Ala Ala Gly Arg Gly Glu Thr Cys Tyr Arg Thr Val Ser Gly Met Asp Gly Met Lys Cys Gly Pro Cys Leu Arg Cys Gln Pro Ser Asn Cys Glu Asp Pro Phe Gly Glu Glu Phe Gly Ile Cys Lys Asp Cys Pro Tyr Gly Thr Phe Gly Met Asp Cys Arg Glu Thr Cys Asn Cys Gln Ser Cys Ile Cys Asp Arg Gly Thr Gly Lys Cys Leu Lys Phe Pro Phe Phe Gln Tyr Ser Val Thr Lys Ser Ser Asn Arg Phe Val Ser Leu Thr Glu His Asp Met Ala Ser Gly Asp Gly Asn Ile Val Arg Glu Glu Val Val Lys Glu Asn Ala Ala Gly Ser Pro Val Met Arg Lys Trp Leu Asn Pro Arg
Another object of the present invention is a peptide composed of between almost 5 and 50 aminoacids and comprising an epitope, the said peptide showing at least a part of the sequence SEQ ID NO2. Such a peptide and proteins can be obtained by expressing oligonucleotides encoding them. Such oligonucleotides, which constitute other objects of the invention, can be mRNA or DNA, and in particular can be oligonucleotides comprising at least 50, preferentially 75 and more preferentially 150 nucleotides of the following sequence SEQ ID NO1.
CTTCCCACCA GCAAAGACCA CGACTGGAGA GCCGAGCCGG AGGCAGCTGG GAAAC ATG AAG AGC GTC TTG CTG CTG ACC ACG CTC CTC GTG CCT GCA CAC CTG GTG GCC GCC TGG AGC AAT AAT TAT GCG GTG GAC TGC CCT CAA CAC TGT GAC AGC AGT GAG TGC AAA AGC AGC CCG CGC TGC AAG AGG ACA GTG CTC GAC GAC TGT GGC TGC TGC CGA GTG TGC GCT GCA GGG CGG GGA GAA ACT TGC TAC CGC ACA GTC TCA GGC ATG GAT GGC ATG AAG TGT GGC CCG GGG CTG AGG TGT CAG CCT TCT AAT GGG GAG GAT CCT TTT GGT GAA GAG TTT GGT ATC TGC AAA GAC TGT CCC TAC GGC ACC TTC GGG ATG GAT TGC AGA GAG ACC TGC AAC TGC CAG TCA GGC ATC TGT GAC AGG GGG ACG GGA AAA TGC CTG AAA TTC CCC TTC TTC CAA TAT TCA GTA ACC AAG TCT TCC AAC AGA TTT GTT TCT CTC ACG GAG CAT GAC ATG GCA TCT GGA GAT GGC AAT ATT GTG AGA GAA GAA GTT GTG AAA GAG AAT GCT GCC GGG TCT CCC GTA ATG AGG AAA TGG TTA AAT CCA CGC T GATCCCGGCT GTGATTTCTG AGAGAAGGCTCTATTTTCGT ATTGTTCAA CACACAGCCA ACATTTTAGG AACTTTCTAG ATATAGCATA AGTACATGTA ATTTTTGAAG ATCCAAATTG TGATGCATGG TGGATCCAGA AAACAAAAAGTAGGATACTT ACAATCCATA ACATCCATAT GACTGAACAC TTGTATGTGT TTGTTAAATATTCGAATGCA TGTAGATTTG TTAAATGTGT GTGTATAGTA ACACTGAAGA ACTAAAAATG CAATTTAGGT AATCTTACAT GGAGACAGGT CAACCAAAGA GGGAGCTAGG CAAAGCTGAA GACCGCAGTG AGTCAAATTA GTTCTTTGAC TTTGATGTAC ATTAATGTTG GGATATGGAA TGAAGACTTA AGAGCAGGAG AAGATGGGGA GGGGGTGGGA GTGGGAAATA AAATATTTAG CCCTTCCTTG GTAGGTAGCT TCTCTAGAAT TTAATTGTGC TTTTTTTTTT TTTTTGGCTT TGGGAAAAGT CAAAATAAAA CAACCAGAAA ACCCCTGAAG GAAGTAAGAT GTTTGAAGCT TATGGAAATT TGAGTAACAA ACAGCTTTGA ACTGAGAGCA ATTTCAAAAG GCTGCTGATG TAGTTCCCGG GTTACCTGTA TCTGAAGGAC GGTTCTGGGG CATAGGAAAC ACATACACTT CCATAAATAG CTTTAACGTA TGCCACCTCA GAGATAAATC TAAGAAGTAT TTTACCCACT GTGGTTTGT GTGTGTATGA AGGTAAATAT TTATATATTT TTATAAATAA ATGTGTTAGT GCAAGTCATC TTCCCTACCC ATATTTATCA TCCTCTTGAG GAAAGAAATC TAGTATTATT TGTTGAAAAT GGTTAGAATA AAAACCTATG ACTCTATAAG GTTTTCAAAC ATCTGAGGCA TGATAAATTT ATTATCCATA ATTATAGGAG TCACTCTGGA TTTCAAAAAA TGTCAAAAAA TGAGCAACAG AGGGACCTTA TTTAAACATA AGTGCTGTGA CTTCGGTGAA TTTTCAATTT AAGGTATGAA AATAAGTTTT TAGGAGGTTT GTAAAAGAAG AATCAATTTT CAGCAGAAAA CATGTCAACT TTAAAATATA GGTGGAATTA GGAGTATATT TGAAAGAATC TTAGCACAAA CAGGACTGTT GTACTAGATG TTCTTAGGAA ATATCTCAGA AGTATTTTAT TTGAAGTGAA GAACTTATTT AAGAATTATT TCAGTATTTA CCTGTATTTT ATTCTTGAAG TTGGCCAACA GAGTTGTGAA TGTGTGTGGA AGGCCTTTGA ATGTAAAGCT GCATAAGCTG TTAGGTTTTG TTTTAAAAGG ACATGTTTAT TATTGTTCAA TAAAAAAGAA CAAGATAC
The proteins or the peptides can be produced by a method comprising:
introducing at least a part of sequence SEQ ID NO1 in an expression vector,
introducing the said vector in a suitable host cell, and
growing the said cell in conditions allowing the expression of the said protein.
The man skilled in the art is able to find without undue experiment which vector and which host cell will be suitable for the expression of the peptides and proteins according to the present invention.
In a preferred embodiment, the said peptides and proteins are produced by introducing a DNA sequence encoding for them according to the present invention, in the pcDNA3 vector. The pcDNA3 vector containing the said DNA is transfected in COS cells and these cells are grown in a suitable complete medium.
The peptides and proteins are recovered by methods which are known by the man skilled in the art.
For carrying out this method and the other embodiments described in the present application, the man skilled in the art may advantageously refer to the following manual: Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor ed. NEW YORK, or one of its more recent editions.
The inventors have shown that the expression of the nucleic acid sequence of SEQ ID O1 encoding ESM-1 in eukaryotic cells, such as endothelial cells and established cell lines transfected with a vector containing the nucleic acid sequence of SEQ ID NO1 led to the secretion of a ESM-1 protein lacking the 19 aminoacids of the signal peptide. Thus, the secreted ESM-1 protein was cleaved at the predicted site, leading to a mature ESM-1 polypeptide of 165 aminoacids having the aminoacid sequence of SEQ ID NO3.
The inventors have also shown that the secreted form of the ESM-1 protein having the aminoacid sequence of SEQ ID NO3 was able to bind to the cell-surface of human peripheral blood mononuclear cells (PBMC) as well as to the cell-surface of cells of the Jurkat cell line which is a B resting lymphoblastoid cell line. The binding of ESM-1 to the Jurkat cells was mostly dependent on the presence of divalent ions. This divalent ion-dependent binding of ESM-1 was found saturable, consistent with a receptor-like structure.
Further, co-immunoprecipitations demonstrated that ESM-1 was solely co-immunoprecipitated with anti-LFA-1 monoclonal antibodies.
The interaction between ESM-1 and LFA-1 was confirmed to be specific.
The interaction of LFA-1 and purified human secreted ESM-1 was examined using a BIAcore Biosensor System in order to monitor both the association and the dissociation between LFA-1 and ESM-1 in a real time. The measures of the rate constants demonstrated a physical interaction between ESM-1 and LFA-1 with a dissociation constant of 18.7 mM, close to that found between soluble ICAM-1 and LFA-1 of 60 nM. From the three LFA-1 monoclonal antibodies tested, two of them are neutralizing, but neither of them was able to inhibit ESM-1 binding. The inventors believe that there was no direct relationship between ESM-1 and the neutralizing activity of LFA-1 monoclonal antibodies, and that the ESM-1 binding site was not close to the sites involved in LFA-1/ICAM-1 interactions, namely the I domain.
In contrast to ICAM-1, the binding of ESM-1 to the Jurkat cells was higher at 4xc2x0 C. than at 37xc2x0 C. Further, the results of ESM-1 binding by various cell lines induced by PMA suggest that the ESM-1 binding site within the LFA-1 molecule is regulated and that among the different conformational or states which occur during the time course of LFA-1 activation, one of the earliest activation state of the LFA-1 molecule should include up-regulation of the ESM-1 binding site.
The inventors results also showed that ESM-1 reduced significantly the binding of soluble 3D-ICAM-1/Fc to Jurkat cells. Inversely, the soluble 3D-ICAM-1/Fc was shown to reduce to binding of ESM-1 to Jurkat cells. These results show that ESM-1 and ICAM-1 compete for the binding to the LFA-1.
Thus, the inventors have shown that the proteoglycan ESM-1, which is secreted by endothelial cells, interacts with LFA-1 and may influence directly LFA-1 function on human Jurkat cell line and also on circulating human lymphocytes and monocytes. ESM-1 is thus implicated in the regulation of leukocyte extravasion at the inflammatory sites, because of the essential role of the ICAM-1/LFA-1 interactions during firm adhesion.
In addition, ESM-1 might modulate the LFA-1/ICAM-1 costimulatory pathway and orientate the Th1/Th2 balance of the immune response, as it has been reported for anti-ICAM-1 and anti-LFA-1 blocking monoclonal antibodies (Zuckerman et al., 1998. Salomon et al., 1998).
In addition, a modification of the circulating level of ESM-1 has been shown in a population of mixed pulmonary of systemic septic patients. In all the groups of patients, the mean value of ESM-1 was highly elevated compared to healthy subjects. This increase in circulating ESM-1 was correlated with the level of clinical illness severity. Highest levels of circulating ESM-1 at the early stage of the disease were associated with a lower probability of survival after ten days.
Further, previous blood markers of sepsis such as CRP, procalcitonin and soluble ICAM-1 were not able to predict the prognosis of death, in contrast to the values obtained for circulating ESM-1. The fact that ESM-1 concentrations in blood can attain more than 100 ng/mL in patients with septic shock, it can be speculated that ESM-1 may have a potent in vivo regulatory activity on ICAM-1 and LFA-1-mediated functions. Thus, ESM-1, and more particularly the secreted form of ESM-1 having the aminoacid sequence of SEQ ID NO3 may be considered as a novel class of natural endothelial cell-derived molecule able to regulate LFA-1/ICAM-1 interactions and probably LFA-1 mediated functions. Thus, ESM-1 is implicated in the regulation of the LFA-1/ICAM-1 pathway and influence the recruitment of circulating lymphocytes to sites of inflammation as well as LFA-1 dependent leukocyte activation.
The peptides and proteins according to the present invention can be used in a method for the therapy of mammalian organisms of diseases linked to the leukocyte migration, wherein the said organisms are treated with these proteins or peptides. In a preferred embodiment, these peptides and proteins can be used in a method for the therapy of inflammatory diseases, in particular those which are chronic.
Such a method can be carried out by administering the said peptides or proteins to mammalian organisms, including man, the said peptides and proteins being in the form of compositions containing a suitable dose of the said peptides and proteins, as well as pharmacologically acceptable excipients.
The compositions containing peptides and proteins as described hereabove constitute other objects of the present invention.
Diseases which can be treated by the method according to the present invention can be in a general manner those which are linked with leukocyte migration, and in particular asthma, inflammatory reaction during sepsis, rhumatoid, vasculitis, or allogenic or xenogenic rejection during transplantation.
The present invention moreover relates to a method of diagnosis of diseases linked to leukocyte migration comprising the following steps:
bringing in contact the sample of the said organism with a protein or a peptide as described hereabove, and
determining the antibody-antigen complexes which are formed.
A further object of the present invention is a method for determining anti-ESM-1 antibodies in a biological sample of an organism comprising the following steps:
bringing in contact a biological sample of an organism with a protein or a peptide as described hereabove, and
determining the complexes which are formed.
The present invention moreover relates to kits for carrying out the said methods comprising at least one of the peptides or proteins as described hereabove.